RNA interference (RNAi) is an extremely versatile tool for inhibition of gene expression. RNAi is based on the introduction of double stranded RNA (dsRNA) molecules into cells, whereby one strand is complementary to the coding region of a target gene. Through pairing of the specific mRNA with the introduced RNA molecule, the mRNA is degraded by a cellular mechanism. Short (30 bp) interfering RNA duplexes (siRNA) have been shown to be effective, and do not provoke an immune response, extending the application to mammalian cells. Small hairpin RNAs (shRNAs) transcribed in vivo, are able to trigger degradation of corresponding mRNAs similar to the siRNAs. Micro RNAs (miRNAs) are the endogenous form of shRNAs that carry out the gene silencing function in vivo.
shRNA expression has been accomplished using gene expression vectors, with RNA polymerase III (Pol III) or Polymerase II (Pol II) promoters, with expression occurring in mice injected with the shRNA expression vectors, however, gene inhibition was temporally and spatially restricted. Moreover stable integration of the construct is not readily accomplished or validated in current systems.